1. Field of the Invention
The present invention relates to immunoassays, methods for carrying out immunoassays, immunoassay kits and methods for manufacturing immunoassay kits. In particular, the invention has relevance to capillary (especially microcapillary) immunoassay technology.
2. Related Art
Immunoassays (IAs) are powerful biochemical tools that allow measurement of the concentration of a substance in a clinical, medical, biotechnological or environmental sample. IAs normally utilise the specific interaction between antibodies and their antigens, and are used to measure biomolecules and small molecules in diverse applications including detection of pathogens, infection, drugs, disease biomarkers, environmental contaminants, biowarfare agents and toxins in food products. Heterogenous IAs work by immobilising an antigen or capture antibody onto, e.g., a plastic surface. The presence of an antigen or antibody in a sample can then be determined by a variety of methods, the most common being to label either antigen or the antibody. Common labels include enzymes, as used in enzyme-linked immunosorbent assay (ELISA). Other labels include colloidal gold (as used in lateral flow assays), radioisotopes such as I-125 as used in radioimmunoassay (RIA), magnetic labels as used in magnetic immunoassay (MIA) and fluorescent labels.
The most common platform for IA in life sciences laboratories are microtitre plates. The experimental procedure for a microtitre plate IA typically starts with coating the microwell surfaces (over an extended period of time, e.g. overnight) with the antigen or capture antibody, followed by vigorous washing. The samples are then added to the wells and incubated for a defined time, typically 2-8 hours for maximum sensitivity. Detection antibodies are then added to the wells after another extensive washing and incubated for more than 1 h. This results in a tedious, long procedure and high consumption of expensive reagents (the minimum operational volumes for 96-well microtitre plate wells are 50-100 μl). Specialised equipment for signal detection (e.g. a microplate reader) is also required, representing an investment cost of up to £20,000.
To address the problems of prolonged incubation times and high consumption of expensive reagents typical of microtitre plate based assays, alternative IA techniques have been developed which use plastic surfaces having a large surface area to volume, such as of the type shown by microbeads or microfluidics devices instead of microtitre plates.
Fluorescent or magnetic microbeads offer a very high specific surface area for the immobilization of antigen or capture antibody and can effectively be multiplexed or automated by means of a robotic system and a flow cytometer for signal detection. Nevertheless, to set up a microbead-based IA normally requires an investment of £50,000-100,000.
IA microbeads offer a very high specific surface area for the immobilization of antigen or capture antibody. Microbead IA offer advantages over microtitre plate IA being more suited to automation by means of a robotic system. A further advantage is the possibility of multiplex analyte measurement whereby one sample is analysed for multiple analytes simultaneously. Specialised equipment is required to detect the signal generated by microbead IA, such as a flow cytometer or a microbead analyzer for signal detection. Therefore the equipment required to set up a microbead-based IA normally requires an investment of £30,000-100,000.
In addition, some recent microfluidic technologies offer the possibility of processing multiple samples via automation, requiring minimum volumes of sample. Various types of microfluidic IA technologies are reviewed in Bange et al 2005.
Yacoub-George et al 2007 disclose a microfluidic apparatus for carrying out immunoassays. Their apparatus includes 10 fused silica capillaries held in a specially-designed cartridge. The cartridge is coupled to microfluidic pumps for individual control of the fluid type and fluid flow provided to each capillary. Different capillary elements have different antibodies immobilised at the internal bore surface. The apparatus also includes a specially-designed light-sensing detector module for measuring the immunoassay results from the capillaries.
However, for many applications, the cost of producing microfluidic devices e.g. by soft lithography with integrated signal detectors remains too high to be cost effective for IAs performed in many life sciences, clinical diagnostic, or other laboratories.
Other known types of IAs employ capillary elements, see for example U.S. Pat. No. 5,624,850. In these assays, the bore of the capillary providing a fluid conduit for a sample and antigen proteins or antibodies being immobilised at the internal surface of the bore of the capillary. Capillary-based IAs provide an advantage in terms of the available surface area of the capillary bore compared with the volume of sample required for microtitre plate based IAs. In addition, the high surface to volume ratios of capillaries compared with microtitre plates means that the length of the incubation times required for e.g. antigen-antibody binding are shortened.
A number of apparatuses for performing capillary-based IAs have been described. For example, U.S. Pat. No. 4,116,638 discloses a device for carrying out IAs using multiple capillaries simultaneously. The device comprises a vial with a circular disc inserted into the vial and openings in the disc into which capillaries can be inserted. In addition, the disc has a larger opening in its centre into which a tube can be inserted. This tube can be used to add e.g. samples to the vial which are then taken up by the capillaries.
Another apparatus for capillary-based immunoassays is described in U.S. Pat. No. 4,883,760. In one example, one or more capillaries are held in a flexible support structure and are initially suspended with their lower ends free. Samples etc. are introduced into the capillaries through an aperture in the support. The capillaries can then be drained by deflecting the upper part of the support downwards until the lower ends of the capillaries touch an absorbent material positioned below them.
U.S. Pat. No. 5,976,896 and U.S. Pat. No. 6,517,778 also describe an apparatus for capillary-based immunoassays. In one example, a cartridge comprising four capillary tubes is used to screen for different analytes in a milk sample. Three of the four capillaries in the cartridge were each coated with a different reagent thereby allowing detection of different analytes in a competitive immunoassay. The fourth capillary was left blank and acted as a control.
U.S. Pat. No. 4,590,157 discloses, in one embodiment, a device formed by connecting several capillary elements in series. Each capillary element is formed of a transparent material. Suitable examples given are glass, polyvinyl chloride or polystyrene. Each capillary element has a length of about 2 cm, an internal bore diameter of about 1 mm and an external diameter of about 2 mm. Each capillary element has different antibodies, antigens or haptenic substances adsorbed or covalently bonded at the surface of the bore. In use, a sample fluid is drawn through the series of capillary elements. The type of immunoassay performed is typically an enzyme-linked immunosorbent assay (ELISA). In an alternative embodiment, three capillary elements are arranged in parallel, each capillary tube being capable of indicating the presence of an analyte (digoxin) within different predetermined concentration ranges. In this way, a quantitative assay is provided. The unknown sample is draw through the parallel capillary elements by aspiration by three corresponding plungers. In each embodiment, the results of the assay are determined by an assessment of the colour change associated with each capillary element. A similar disclosure is provided by Healey et al, 1983.
Different methods for detecting signals produced in capillary-based IAs have also been described. For example, U.S. Pat. No. 4,716,121 describes a capillary-based fluorescent immunoassay in which an optical fibre is inserted into the capillary. Illumination of the fibre results in an evanescent wave being produced in the sample within the capillary which in turn excites fluorescently-tagged complexes. The resulting fluorescence then enters the fibre and is collected by a fluorimeter.
Capillaries have also been used as measuring devices in immunoassays. For example, U.S. Pat. No. 4,454,235 describes an apparatus for performing immunoassays in which a capillary is used to transfer a precise amount from a first container containing a mixture of sample and fluorogenic agent to a second container containing a second reagent. The capillary tube in this case is held within a support so that at least one end of the capillary is accessible to fluid. Once the mixture has been transferred to the second container, fluorescence is measured by placing the second container in a fluorometer.
Another system for carrying out IAs is described in U.S. Pat. No. 6,340,598. In this system a biosensor comprising a planar waveguide is used to detect the presence of an analyte in a sample. The waveguide in this case forms at least one wall of the sample reservoir and a light source is position to focus light into the waveguide, wherein internal reflection within the waveguide leads to the production of evanescent light. The apparatus further has a detector for detecting fluorescence emitted by tracer molecules in a test solution in response to stimulation with evanescent light.